Signaling Pathways Used by the Specialized Pro-Resolving Mediator Maresin 2 Regulate Goblet Cell Function: Comparison with Maresin 1

Specialized pro-resolving mediators (SPMs), including Maresins (MaR)-1 and 2, contribute to tear film homeostasis and resolve conjunctival inflammation. We investigated MaR2′s signaling pathways in goblet cells (GC) from rat conjunctiva. Agonist-induced [Ca2+]i and high-molecular weight glycoconjugate secretion were measured. MaR2 increased [Ca2+]i and stimulated secretion. MaR2 and MaR1 stimulate conjunctival goblet cell function, especially secretion, by activating different but overlapping GPCR and signaling pathways, and furthermore counter-regulate histamine stimulated increase in [Ca2+]i. Thus, MaR2 and MaR1 play a role in maintaining the ocular surface and tear film homeostasis in health and disease. As MaR2 and MaR1 modulate conjunctival goblet cell function, they each may have potential as novel, but differing, options for the treatment of ocular surface inflammatory diseases including allergic conjunctivitis and dry eye disease. We conclude that in conjunctival GC MaR2 and MaR1, both increase the [Ca2+]i and stimulate secretion to maintain homeostasis by using one set of different, but overlapping, signaling pathways to increase [Ca2+]i and another set to stimulate secretion. MaR2 also resolves ocular allergy.


Introduction
The ocular surface including the cornea and conjunctiva is covered by a protective tear film. The innermost layer of the tear film is the mucous layer, which consists of electrolytes, water, and mucins produced by conjunctival goblet cells [1]. The mucins provide a critical line of defense from the external environment and play a role in maintaining health [2]. A disturbance in the homeostasis of mucin secretion has been described in a variety of inflammatory ocular surface diseases, including allergic conjunctivitis, Sjogren's syndrome, and dry eye disease [2][3][4][5][6][7]. Disruption to mucin amount, structure, or hydration is deleterious to corneal clarity and hence vision. The resolution of inflammation is an active process with the production of pro-resolution mediators [8]. A group of lipid mediators called specialized pro-resolving mediators (SPMs), including the Maresins (MaRs), maintain homeostasis and counter regulate pro-inflammatory mediators in disease [9,10].
MaR1 and MaR2 are biosynthesized in macrophages and other tissues and are derived from the endogenous ω-3 fatty acid docosahexaenoic acid (DHA) [11]. MaR1 and MaR2 are synthesized through multiple enzymatic steps. Biosynthesis is initiated by 12-lipoxygenase , which converts DHA to 14-hydroperoxydocosahexaenoic acid. The two MaRs share a biosynthesis pathway until 13S, 14S-epoxy-maresin. 13S, 14S-epoxy-maresin is enzymatically converted to MaR1 by a hydrolase and to MaR2 by a soluble epoxide hydrolase [11,12]. Both MaRs consist of a carbon chain which is 22 carbons long, a carboxyl group, two hydroxyl groups, and six double bonds; however, the placement of the hydroxyl groups and double bonds are dissimilar.
MaR1 and MaR2 function by limiting polymorphonuclear (PMN) infiltration and stimulating macrophage phagocytosis. By reducing the number of PMNs and removing apoptotic and necrotic cells the MaRs act to resolve inflammation [11,12]. In addition to pro-resolving effects, MaR1 stimulates regeneration and reduces pain. After surgical decapitation of planaria, MaR1 is biosynthesized which accelerates regeneration [13]. MaR1 reduces inflammatory and neuropathic pain by inhibition of TRPV1 [13]. Furthermore, MaR1 is present in human lymphoid tissue (spleen and lymph nodes) and human serum, indicating a possible role in the immune system [14]. Recent investigation of the actions of MaR1 on rat conjunctival goblet cells demonstrated that MaR1 increases [Ca 2+ ] i and stimulates glycoprotein secretion. MaR1 increased [Ca 2+ ] i and stimulated glycoprotein secretion by activating PLC and its downstream effectors, IP 3 , PKC, and by activation of PLD, Ca 2+ -calmodulin kinase (CaMK) II and extracellular regulated kinase (ERK) 1/2 [15].
In the present study, we investigated the action of MaR2 on cultured rat conjunctival goblet cells. To activate goblet cells and stimulate mucin secretion, one of the main signals is an increase in the intracellular Ca 2+ concentration ( [Ca 2+ ] i ) that triggers mucin secretion. Therefore, measuring the [Ca 2+ ] i was used as functional readout in the study presented in this manuscript. We identified the intracellular pathways MaR2 uses by measuring [Ca 2+ ] i and high molecular weight glycoprotein secretion including mucin secretion. We used pharmacologic inhibitors of different signaling pathways followed by addition of MaR2. Furthermore, the effect of MaR2 on histamine was investigated, because the MaRs are thought to play a central role in allergy [15]. MaR1 was used as a control for comparison.
The results from Figure 2 suggest that MaR2 increases [Ca 2+ ] i and this increase leads to secretion.

Maresin 2 Activates the BLT1 Receptor, but Not the ALX/FPR 2 Receptor to Increase [Ca 2+ ] i
The formyl peptide receptors (FPRs) are a family of receptors including three subtypes in mammals; FPR1, FPR2 and FPR3. The first described ligand of the receptor was a formylated peptide from Escherichia coli, which binds with high affinity [21]. The Lipoxinreceptor (ALX)/N-formyl-peptide receptor (FPR2) (ALX/FPR2)-receptor is a complex G-protein coupled receptor (GPCR) which is known to bind a variety of ligands, including proteins/peptides such as Annexin A1 and serum amyloid a (SAA), lipids like RvD1 and LxA 4 and small molecules like compound 43 (C43) [19,22]. The ALX/FPR2 receptor is present in rat conjunctival goblet cells, confirmed both by western blot analysis and RT-PCR [19,[23][24][25]. Annexin A1 (AnxA1), LxA4 and RvD1 have been found to increase [Ca 2+ ] i and stimulate secretion by binding to the receptor in rat conjunctival goblet cells [19,26,27]. Moreover, MaR1 is dependent on the ALX/FPR-2 receptor to increase [Ca 2+ ] i and to stimulate glycoconjugate secretion in conjunctival goblet cells [15]. Thus, we determined the role of the ALX/FPR2-receptor in MaR2-stimulated increase in [Ca 2+ ] i .
To explore the role of the ALX/FPR2-receptor in glycoconjugate secretion, goblet cells were incubated with BOC2 for 30 min prior to stimulation with MaR2 and the controls MaR1 and LXA 4 . MaR2 increased secretion 1.8 ± 0.3 -fold above basal (Figure 3b; n = 4). The MaR2 response was not significantly inhibited by BOC2, while secretion stimulated by the positive controls MaR1 was decreased and LXA 4 was significantly inhibited by the ALX/FPR2 inhibitor. These data indicate that MaR2 does not utilize the ALX/FPR2receptor to increase [Ca 2+ ] i or stimulate secretion. LTB 4 activates the GPCR receptor, BLT1, to cause chemotactic, pro-inflammatory actions [28]. SPMs including RvE1 and MaR1 also bind to this receptor, but are proresolving [15,29]. To examine if MaR2 is using the BLT1 receptor to increase [Ca 2+ ] i , rat conjunctival goblet cells were treated with an inhibitor of the BLT1 receptor, U-75302 To examine the dependency of MaR2-stimulate secretion on the BLT1 receptor, rat conjunctival goblet cells were treated with U-75302 (10 −6 M) for 30 min before addition of MaR2. MaR2 increased secretion 1.8 ± 0.3 -fold above basal (Figure 3d; n = 4). MaR2 was not significantly inhibited by U-75302. Secretion stimulated by the positive controls MaR1 and LTB 4 was significantly inhibited by U-75302. These data indicate that MaR2 uses the BLT1-receptor to increase [Ca 2+ ] i , but not secretion.
To examine the dependency of MaR2-stimulate secretion on the BLT1 receptor, rat conjunctival goblet cells were treated with U-75302 (10 −6 M) for 30 min before addition of MaR2. MaR2 increased secretion 1.8 ± 0.3 -fold above basal (Figure 3d; n = 4). MaR2 was not significantly inhibited by U-75302. Secretion stimulated by the positive controls MaR1 and LTB4 was significantly inhibited by U-75302. These data indicate that MaR2 uses the BLT1-receptor to increase [Ca 2+ ]i, but not secretion.   These results show that when MaR2 activates its receptor first, MaR1 can also activate it, suggesting that MaR2 and MaR1 are activating different receptors, or overlapping areas on the same receptor. In contrast, when MaR1 activates its receptor first, MaR2 cannot activate it, suggesting that MaR1 and MaR2 activate the same receptor.  These results show that when MaR2 activates its receptor first, MaR1 can also activate it, suggesting that MaR2 and MaR1 are activating different receptors, or overlapping areas on the same receptor. In contrast, when MaR1 activates its receptor first, MaR2 cannot activate it, suggesting that MaR1 and MaR2 activate the same receptor.

Maresin 2 Increase in [Ca 2+ ] i , but Not Secretion , Is Independent of the PLC-Pathway in Rat Conjunctival Goblet Cells
The PLC pathway is activated in a variety of cellular processes, including exocytosis and fluid secretion [30]. This intracellular signaling pathway is essential for the function of MaR1 in rat conjunctival goblet cells [15]. To determine if MaR2 uses the same pathway components to increase [Ca 2+ Figure 5a; n = 4). Treatment with U-73122 or U-73343 followed by MaR2 caused an increase in peak [Ca 2+ ] i to 99.28 ± 36.77 (p = 0.072), and 118.95 ± 47.13 (p = 0.20), respectively, that were unchanged when compared to MaR2 stimulation. MaR1 and Cch stimulation, in contrast to that of MaR2, is dependent on PLC to increase [Ca 2+ ] i as their action on [Ca 2+ ] i was decreased by U-73122, but not by U-73343 ( Figure 5a; n = 4).
The action of PLC on MaR2-stimulated increase in glycoconjugate secretion was next investigated. MaR2 increased secretion 1.9 ± 0.1 -fold above basal ( Figure 5b; n = 4). The response was significantly blocked by U73122 to 0.9 ± 0.2 (p = 0.003), but not by the inactive control U73343 (3.6 ± 2.1) (p = 0.44). MaR1-and Cch-stimulated increase in secretion were also significantly blocked by U73122, but not by U73343 (Figure 5b; n = 4). We conclude that MaR2 is dependent upon activation of the PLC pathway to stimulate glycoprotein secretion, but MaR2 does not use PLC to increase [Ca 2+ ] i .
Activation of the PLC pathway produces IP 3 which binds to its intracellular receptor on the ER causing release of Ca 2+ from intracellular calcium stores increasing [Ca 2+ ] i . To determine if MaR2 is dependent of the downstream molecules that activation of the PLC pathway produces, cells were treated with the IP 3 (Figure 5c; n = 5). We conclude that the action of MaR2 is independent of the action of IP 3 on its receptor to increase [Ca 2+ The effect 2APB on MaR2-stimulated increase in glycoconjugate secretion was then explored. MaR2-increased secretion was 3.3 ± 0.5 -fold above basal (Figure 5d; n = 6). 2APB significantly decreased MaR2-stimulated response to 1.3 ± 0.2 -fold (p = 0.0002, Figure 5d; n = 6). The action of the positive control, Cch, on secretion was also significantly inhibited by 2APB (Figure 5d; n = 6). We conclude that the action of MaR2 on [Ca 2+ ] i, but not secretion, is independent of the action of IP 3 on its receptor to increase [Ca 2+ To determine if MaR2 is using intracellular calcium stores to increase [Ca 2+ ] by other mechanisms than PLC-IP 3 pathway, we used the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) inhibitor thapsigargin. Thapsigargin blocks the uptake of Ca 2+ into intracellular stores so that the cytoplasmic [Ca 2+ ] i increases by a passive leak from the ER. If an agonist uses the same intracellular Ca 2+ store as thapsigargin, the increase in [Ca 2+ ] i by an agonist added after thapsigargin will be decreased. Conjunctival goblet cells were treated by thapsigargin (10 −5 M) for 15 min that releases Ca 2+ from the intracellular stores and then stimulated with MaR2 (10 −8 M), or the positive controls MaR1 (10 −8 M) or Cch (10 −4 M). MaR2 increased [Ca 2+ ] i to a peak of 219.10 ± 21.63 nM (p = 0.00053, Figure 5e,g; n = 3). Treatment with thapsigargin caused a MaR2 increase in [Ca 2+ ] i to a peak of 85.68 ± 4.02 nM that was significantly decreased compared to MaR2 alone (p = 0.0037, Figure 5f,g; n = 3). A similar effect of thapsigargin was detected using MaR1 and Cch. Thus, MaR2 is dependent on a release of Ca 2+ from intracellular calcium stores to increase [Ca 2+ ] i . n = 3). A similar effect of thapsigargin was detected using MaR1 and Cch. Thus, MaR2 is dependent on a release of Ca 2+ from intracellular calcium stores to increase [Ca 2+ ]i.
MaR2 does not use PLC to increase [Ca 2+ ]i as neither the active PLC inhibitor nor the inhibitor of IP3 for its receptor on the ER blocked MaR2 increase in [Ca 2+ ]i. MaR2, however, does appear to use PLC to stimulate secretion as the inhibitors tested are in agreement on their actions. The inhibitor of SERCA on the ER does block MaR2 increase in [Ca 2+ ]i, but this could belong to an as yet unidentified pathway. White triangles indicate individual data points. * shows significance above basal. # shows significance between MaR2 and inhibitor then MaR2 or control and inhibitor then control. MaR2 does not use PLC to increase [Ca 2+ ] i as neither the active PLC inhibitor nor the inhibitor of IP 3 for its receptor on the ER blocked MaR2 increase in [Ca 2+ ] i. MaR2, however, does appear to use PLC to stimulate secretion as the inhibitors tested are in agreement on their actions. The inhibitor of SERCA on the ER does block MaR2 increase in [Ca 2+ ] i, but this could belong to an as yet unidentified pathway.

Maresin 2 Stimulated Increase in [Ca 2+ ] i Is Independent of Extracellular Ca 2+ in Rat Conjunctival Goblet Cells
In conjunctival goblet cells MaR1 is not dependent on extracellular Ca 2+ to increase [Ca 2+ ] i [15]. To determine if MaR2 is dependent on influx of extracellular Ca 2+ , we incu-bated rat conjunctival goblet cells in vehicle with or without CaCl 2 (1.0 mM). Supplementary Figure S3 indicates changes in [Ca 2+ ] i over time. MaR2 with CaCl 2 increased [Ca 2+ ] i to a peak of 95.66 ± 28.34 nM (p = 0.015, Figure 6; n = 4), while MaR2 without CaCl 2 increased [Ca 2+ ] i to a peak of 64.74 ± 15.25 nM, a non-significant decrease (p = 0.37). A similar finding was detected for MaR1 (10 −8 M) (Figure 6; n = 4). In contrast [Ca 2+ ] i stimulated by Cch (10 −4 M) was significantly decreased in the absence of extracellular Ca 2+ (Figure 6; n = 4). We conclude that MaR2, similarly to MaR1, is independent of influx of extracellular Ca 2+ to increase [Ca 2+ ] i , but Cch is not. [Ca 2+ ]i Is Independent of Extracellular Ca 2+ in Rat

Maresin 2 Has Different Dependency on Phospholipase D (PLD) Compared to Phospholipase A2 (PLA2) to Increase [Ca 2+ ]i and Stimulate Secretion in Rat Conjunctival Goblet Cells
Activation of Phospholipase D (PLD) is controlled by multiple mechanisms, activates distinct pathways and is important in cellular functioning [31]. To explore if MaR2 uses PLD to increase [Ca 2+ ]i we used the PLD-inhibitor 1-butanol (1-but) at 0.3% and the inactive control t-butanol (t-but) at 0.3%. Supplementary Figure S5 [Ca 2+ ]i to a peak of 97.89 ± 6.41 nM that was significantly decreased compare to MaR2 alone (p = 0.040). MaR2 (10 −8 M) added after t-butanol (inactive analog) increased [Ca 2+ ]i to a peak of 72.40 ± 17.05 nM that was significantly decreased from MaR2 alone (p = 0.033). For the positive controls, MaR1 and carbachol stimulation of peak in [Ca 2+ ]i was decreased by 1butanol, but not by t-butanol (Figure 8a; n = 3). Although the peak in [Ca 2+ ]i was reduced by 1-butanol, we cannot definitively conclude that the action of MaR2 is dependent on PLD, due to inhibition by the inactive control t-butanol. In contrast, MaR1 and Cch stimulation were dependent on the activation of PLD.
To examine if MaR2 is dependent on PLA2 to increase [Ca 2+ ]i we used the PLA2-inhibitor Aristolochic Acid (AA). Supplementary Figure S5

Maresin 2 Has Different Dependency on Phospholipase D (PLD) Compared to Phospholipase A 2 (PLA 2 ) to Increase [Ca 2+ ] i and Stimulate Secretion in Rat Conjunctival Goblet Cells
Activation of Phospholipase D (PLD) is controlled by multiple mechanisms, activates distinct pathways and is important in cellular functioning [31]. To explore if MaR2 uses PLD to increase [Ca 2+ ] i we used the PLD-inhibitor 1-butanol (1-but) at 0.3% and the inactive control t-butanol (t-but) at 0.3%. Supplementary Figure S5 [Ca 2+ ] i to a peak of 97.89 ± 6.41 nM that was significantly decreased compare to MaR2 alone (p = 0.040). MaR2 (10 −8 M) added after t-butanol (inactive analog) increased [Ca 2+ ] i to a peak of 72.40 ± 17.05 nM that was significantly decreased from MaR2 alone (p = 0.033). For the positive controls, MaR1 and carbachol stimulation of peak in [Ca 2+ ] i was decreased by 1-butanol, but not by t-butanol (Figure 8a; n = 3). Although the peak in [Ca 2+ ] i was reduced by 1-butanol, we cannot definitively conclude that the action of MaR2 is dependent on PLD, due to inhibition by the inactive control t-butanol. In contrast, MaR1 and Cch stimulation were dependent on the activation of PLD.
To examine if MaR2 is dependent on PLA 2 to increase [Ca 2+ ] i we used the PLA 2inhibitor Aristolochic Acid (AA). Supplementary Figure S5 indicates changes in [Ca 2+ ] i over time. MaR2 (10 −8 M) caused an increase in [Ca 2+ ] i to a peak of 301.53 ± 17.7 nM (p = 0.05, Figure 8c; n = 4). When incubated with AA 10 −5 M or AA 10 −6 M, MaR2 caused an increase in [Ca 2+ ] i to a peak of 108.77 ± 14.83 nM or 129.09 ± 25.71 nM, respectively values that were significantly decreased from MaR2 alone (p < 0.001 for AA 10 −5 M and p = 0.001 for AA 10 −6 M). The action of Cch on peak increase in [Ca 2+ ] i was blocked by AA at 10 −5 M (Figure 8c; n= 4). Thus MaR2 activates PLA2 to increase [Ca 2+ ] i . 1.7 ± 0.2 -fold above basal (p = 0.003, Figure 8d; n=6). When incubated with AA at 10 −5 M MaR2 increased secretion to 1.7 ± 0.8 -fold above basal (p = 0.97), not a significantly different value from stimulation with MaR2 alone. The increase in glycoconjugate secretion stimulated by the positive control, Cch was significantly decreased by AA (Figure 8d; n =  6). This indicates that MaR2 uses PLA2 to increase [Ca 2+ ]i, but not to stimulate glycoconjugate secretion. White triangles indicate individual data points. * shows significance above basal. # shows significance between agonist and inhibitor followed by agonist.

Maresin 2 Uses Protein Kinase A to Increase [Ca 2+ ]i and Stimulate Secretion in Rat Conjunctival Goblet Cells
When a ligand activates Gαs, adenylyl cyclase (AC) catalyzes ATP to cAMP that in turn stimulates the activity of cAMP dependent protein kinase A (PKA). This is one To determine if the action of MaR2 is dependent on PLA 2 to stimulate glycoconjugate secretion, conjunctival goblet cells were preincubated with AA. MaR2 increased secretion 1.7 ± 0.2 -fold above basal (p = 0.003, Figure 8d; n=6). When incubated with AA at 10 −5 M MaR2 increased secretion to 1.7 ± 0.8 -fold above basal (p = 0.97), not a significantly different value from stimulation with MaR2 alone. The increase in glycoconjugate secretion stimulated by the positive control, Cch was significantly decreased by AA (Figure 8d; n = 6). This indicates that MaR2 uses PLA 2 to increase [Ca 2+ ] i , but not to stimulate glycoconjugate secretion.

Maresin 2 Uses Protein Kinase A to Increase [Ca 2+ ] i and Stimulate Secretion in Rat Conjunctival Goblet Cells
When a ligand activates Gαs, adenylyl cyclase (AC) catalyzes ATP to cAMP that in turn stimulates the activity of cAMP dependent protein kinase A (PKA). This is one among a variety of functions of cAMP [32]. To explore if MaR2 uses PKA, we incubated rat conjunctival goblet cells with the PKA-inhibitor H89 ( Figure 9a; n = 5). Incubation with H89 increased [Ca 2+ ] i to a peak of 40.66 ± 1.94 nM that was different from MaR2 alone (p = 0.023). The action of MaR1 was not inhibited by H89, but of VIP was blocked (Figure 9a; n = 5). This indicates that MaR2 activates PKA to increase [Ca 2+ ] i . peak of 91.27 ± 17.97 nM (p = 0.00010, Figure 9a; n = 5). Incubation with H89 increased [Ca 2+ ]i to a peak of 40.66 ± 1.94 nM that was different from MaR2 alone (p = 0.023). The action of MaR1 was not inhibited by H89, but of VIP was blocked (Figure 9a; n = 5). This indicates that MaR2 activates PKA to increase [Ca 2+ ]i.
To determine the dependency of MaR2 on PKA to stimulate glycoconjugate secretion, conjunctival goblet cells were incubated with H89 (10 −5 M) 30 min prior to addition of MaR2. MaR2 stimulated secretion to 3.4 ± 0.5 -fold above basal (p = 2.2 × 10 −5 , Figure 9b; n = 6). Incubation with H89 significantly decreased MaR2-stimulated secretion to 1.8 ± 0.3fold above basal (p=0.004). Secretion stimulated by the positive control VIP was also significantly inhibited by H89. These data indicate that MaR2, but not MaR1, is dependent on activation of PKA to increase [Ca 2+ ]i and glycoconjugate secretion. To determine the dependency of MaR2 on PKA to stimulate glycoconjugate secretion, conjunctival goblet cells were incubated with H89 (10 −5 M) 30 min prior to addition of MaR2. MaR2 stimulated secretion to 3.4 ± 0.5 -fold above basal (p = 2.2 × 10 −5 , Figure 9b; n = 6). Incubation with H89 significantly decreased MaR2-stimulated secretion to 1.8 ± 0.3 -fold above basal (p=0.004). Secretion stimulated by the positive control VIP was also significantly inhibited by H89. These data indicate that MaR2, but not MaR1, is dependent on activation of PKA to increase [Ca 2+ ] i and glycoconjugate secretion.
The leukotriene LTB 4 is a chemoattractant involved in inflammation and immune response and activates inflammatory cells [28]. LTB 4 binds to the BLT1 receptor and to the ALX/FPR2-receptor [35]. To determine if MaR2 and MaR1 act on LTB 4 -stimulated increase in [Ca 2+ ] i , we pre-incubated rat conjunctival goblet cells with MaR2 (10 −8 M) or MaR1 (10 −8 M) for 30 min, then stimulated with LTB 4 (10 −9 M). Supplementary Figure  S7 indicates changes in [Ca 2+ ] i over time. LTB 4 caused an increase in [Ca 2+ ] i to a peak of 128.71 ± 27.51 nM (p=0.0034, Figure 10c; n=4). Incubation with MaR2 caused a LTB 4stimulated increase in [Ca 2+ ] i to a peak of 89.62 ± 31.53 nM (p=0.39) that was not different from stimulation by LTB 4 alone. Incubation with MaR1 significantly decreased the LTB 4stimulated increase in [Ca 2+ ] i to a peak of 47.65 ± 7.65 nM (p=0.030). We conclude that MaR2 does not inhibit LTB 4 -stimulated increase in [Ca 2+ ] i , while MaR1 does.

Discussion
In the present study we showed that MaR2 activates rat conjunctival goblet cells through an increase in [Ca 2+ ] i that stimulates secretion and blocks overproduction of mucin stimulated by histamine, an allergic mediator. Both of these actions are used by MaR2 to maintain homeostasis in both health and disease ( Figure 11). MaR2 uses the BLT1 receptor to increase [Ca 2+ ] i by activation of the cAMP-dependent PKA, PLD, PLC-PKC, and PLA 2 , but not the PLC-IP3, signaling pathways. None of the inhibitors of the signaling components, however, blocked MaR2-stimulated increase in [Ca 2+ ] i completely, indicating that multiple pathways/receptors could be involved in cellular activation. Pre-incubation with thapsigargin completely decreased MaR2-stimulated [Ca 2+ ] i increase, suggesting that activated signaling pathways caused a release of Ca 2+ from intracellular calcium stores. Similarly to other SPMs, such as MaR1, LXA 4 , RvD1, RvD2, and RvE1, MaR2 regulates [Ca 2+ ] i and secretion including MUC5AC in rat conjunctival goblet cells [15,[17][18][19]36]. These actions likely contribute to optimal tear film function under normal, physiological conditions. MaR2 also prevents the overproduction of mucin stimulated by histamine in ocular allergy. Thus MaR2 maintains homeostasis of tear film mucin in both health and disease.
Although being of similar chemical structure, MaR2 and MaR1 activate different receptors. MaR2 to date only uses the BLT1 receptor and uses it only to increase [Ca 2+ ] i . We found that MaR1 uses the BLT1-and the ALX/FPR2 receptor to increase [Ca 2+ ] i , but only the BLT1 receptor to stimulate secretion. Treatment with MaR1 desensitizes MaR2, while preincubation with MaR2 does not affect the MaR1 response. There are several possible mechanisms that might explain how MaR1 attenuates MaR2 response. A possible mechanism of inhibition of MaR2 actions by MaR1 is through activation of the ALX/FPR2 receptor or through other receptors, including the newly identified LGR6 receptor for MaR1 that MaR2 does not stimulate [37]. It should be noted, however, that LGR6 was found in human, but not yet in rat, tissue. Furthermore, MaR1 may attenuate MaR2 by interacting with an overlapping or different region of the BLT1 receptor than MaR2 binds to. To support this hypothesis, we found that MaR1 decreases LTB 4 induced increase in [Ca 2+ ] i , while MaR2 does not. We suggest that MaR1, but not MaR2, may attenuate LTB 4 -and MaR2-dependent BLT1 responses by activating a protein kinase that phosphorylates the BLT1 receptor and counter-regulates it. MaR1, but not MaR2, could contribute to resolution of leukotriene-stimulated inflammation in ocular surface disease.
The BLT1 receptor is activated by the pro-inflammatory chemoattractant LTB 4 [38]. We found that MaR1 was dependent on the BLT-1 receptor to increase [Ca 2+ ] i , and to stimulate glycoconjugate secretion, while MaR2 was only dependent on the BLT1 receptor to increase [Ca 2+ ] i . The fact that the pro-inflammatory mediator LTB 4 and the pro-resolving mediators MaR1 and MaR2 are using the same receptor is an example of biased agonism. Biased agonism is when different ligands bind to a receptor to activate different signal transduction pathways, a phenomenon also found in receptors such as ALX/FPR2 [39]. The BLT1 receptor is a GPCR primarily known to couple to the inhibitory protein of the adenylyl cyclase, G i , and the stimulatory protein G q , the latter of which activates PLC, ultimately inducing chemotaxis [40]. We found that MaR2 can activate the BLT1 receptor, while also increase cAMP levels and stimulate PKA, which are activated by the protein G s . BLT1 does not couple to G s and does not activate adenylyl cyclase suggesting that MaR2 could activate another receptor in rat conjunctival goblet cells to perform its actions. In support of this suggestion another SPM derived from DHA, RvE1, is known to bind to both the ChemR23 and BLT1 receptors. A central role for BLT1 in SPM functioning in rat conjunctival goblet cells is emerging [29].
The BLT1 receptor is activated by the pro-inflammatory chemoattractant LTB4 [38]. We found that MaR1 was dependent on the BLT-1 receptor to increase [Ca 2+ ]i, and to stimulate glycoconjugate secretion, while MaR2 was only dependent on the BLT1 receptor to increase [Ca 2+ ]i. The fact that the pro-inflammatory mediator LTB4 and the pro-resolving mediators MaR1 and MaR2 are using the same receptor is an example of biased agonism. Biased agonism is when different ligands bind to a receptor to activate different signal transduction pathways, a phenomenon also found in receptors such as ALX/FPR2 [39]. The BLT1 receptor is a GPCR primarily known to couple to the inhibitory protein of the adenylyl cyclase, Gi, and the stimulatory protein Gq, the latter of which activates PLC, ultimately inducing chemotaxis [40]. We found that MaR2 can activate the BLT1 receptor, while also increase cAMP levels and stimulate PKA, which are activated by the protein Gs. BLT1 does not couple to Gs and does not activate adenylyl cyclase suggesting that MaR2 could activate another receptor in rat conjunctival goblet cells to perform its actions. In support of this suggestion another SPM derived from DHA, RvE1, is known to bind to both the ChemR23 and BLT1 receptors. A central role for BLT1 in SPM functioning in rat conjunctival goblet cells is emerging [29]. Figure 11. Schematic diagram of signaling pathways activated by Maresin 2 (MaR2) (orange arrows) compared to the pathways activated by Maresin 1 (MaR1) (blue arrows). MaR2 activates the BLT1 receptor activating PLD, AC, PLA2, and PLC. PLD and AC activate downstream molecules that increase [Ca 2+ ]i causing glycoprotein secretion. PLA2 and the PLC pathway stimulates glycoprotein secretion by another unknown mechanism than increasing [Ca 2+ ]i. Consistent with MaR2 and MaR1, interacting with different receptors or different sites on the same receptor, these SPMs differ in the use of the cAMP/PKA signaling pathway. MaR2, but not MaR1, increases cAMP levels and activates PKA to increase [Ca 2+ ]i and stimulate secretion. The only other SPM published to date in rats to use cAMP and PKA to increase in [Ca 2+ ]i and stimulate secretion is RvD2 [36]. Interestingly, in human immune cells MaR1 uses LGR6 to increase cAMP levels and activate PKA. RvD2 in rat and human conjunctival goblet cells uses the GPR18 receptor that activates adenylyl cyclase, to increase cAMP levels and activate PKA. Activation of PKA by itself stimulates secretion, but also increases [Ca 2+ ]i by interacting with the IP3 receptors on intracellular

ALX/FPR2
BLT-1 H1-H4  Figure 11. Schematic diagram of signaling pathways activated by Maresin 2 (MaR2) (orange arrows) compared to the pathways activated by Maresin 1 (MaR1) (blue arrows). MaR2 activates the BLT1 receptor activating PLD, AC, PLA 2, and PLC. PLD and AC activate downstream molecules that increase [Ca 2+ ] i causing glycoprotein secretion. PLA 2 and the PLC pathway stimulates glycoprotein secretion by another unknown mechanism than increasing [Ca 2+ ] i . Consistent with MaR2 and MaR1, interacting with different receptors or different sites on the same receptor, these SPMs differ in the use of the cAMP/PKA signaling pathway. MaR2, but not MaR1, increases cAMP levels and activates PKA to increase [Ca 2+ ] i and stimulate secretion. The only other SPM published to date in rats to use cAMP and PKA to increase in [Ca 2+ ] i and stimulate secretion is RvD2 [36]. Interestingly, in human immune cells MaR1 uses LGR6 to increase cAMP levels and activate PKA. RvD2 in rat and human conjunctival goblet cells uses the GPR18 receptor that activates adenylyl cyclase, to increase cAMP levels and activate PKA. Activation of PKA by itself stimulates secretion, but also increases [Ca 2+ ] i by interacting with the IP 3 receptors on intracellular Ca 2+ stores, likely on endoplasmic reticulum. Vasoactive intestinal peptide (VIP) is a parasympathetic neurotransmitter that like MaR2 and RvD2 stimulates PKA [41]. VIP activates the VPAC1 and the VPAC2 receptors causing activation of adenylyl cyclase that increases levels of cAMP, ultimately activating PKA. The activated PKA increases [Ca 2+ ] i through a mechanism that is similar to that used by RvD2 and slightly different from that used by MaR2. The difference is that VIP and RvD2 stimulate PLC activity to produce IP 3 . IP 3 then binds with its receptors on the ER to release Ca 2+ and cAMP that interacts with the IP 3 receptors to increase Ca 2+ . In contrast, MaR2 does not activate PLC to produce IP 3. Thus IP3 receptors are not involved in the action of MaR2. MaR2 activation of PKA would then increase [Ca 2+ ] i by a different mechanism than RvD2. Further studies are warranted to determine the specifics of the MaR2 cAMP-dependent actions and to compare them with those of RvD2 and VIP.
In spite of MaR2 and MaR1 interacting with different receptors and activating different signaling pathways, MaR2 and MaR1 both use several similar Ca 2+ -dependent signaling pathways. First, both agonists increase [Ca 2+ ] i by release of intracellular Ca 2+ stores, confirmed by inhibition of secretion when the [Ca 2+ ] i was decreased by the Ca 2+ chelator BATPA/AM and when MaR2 and MaR1 stimulated increase in [Ca 2+ ] i was blocked by the SERCA inhibitor thapsigargin that depletes intracellular Ca 2+ stores [15]. There are three main signaling pathways that SPMs use to increase [Ca 2+ ] i and stimulate secretion in conjunctival goblet cells PLA 2 , PLD and PLC. Neither MaR2 nor MaR1 activate PLA 2 to increase [Ca 2+ ] i but MaR2 uses it to stimulate secretion. Both MaR2 and MaR1 activate PLD to increase [Ca 2+ ] i and stimulate secretion, although the negative control for MaR2 and PLD increase in [Ca 2+ ] i was also inhibitory. MaR2 and MaR1 both activate components of the PLC pathway. MaR2 and MaR1 activate PLC, but only MaR1 uses the downstream molecule IP 3 R and only MaR2-stimulated secretion is dependent on these components. Both MaR2 and MaR1 activated PKC to increase [Ca 2+ ] i and stimulate secretion. Surprisingly, both MaR2-and MaR1-stimulated increase in [Ca 2+ ] i are independent of extracellular Ca 2+ . Thus, the PLC pathway has some differences between MaR2 and MaR1 activation, especially in the targets of PLC activation. Whereas MaR1 stimulates the increase in [Ca 2+ ] i and secretion by the well-known PLC pathway that produces IP 3 that releases Ca 2+ from intracellular stores and produces DAG to activate PKC to stimulate secretion, MaR2 only uses these processes to stimulate secretion. As MaR2 and MaR1 activate the PLD pathway, they could use PLD to activate PKC via an increase in Ca 2+ . In contrast, MaR2 does not use PLC to increase Ca 2+ and activate PKC, while MaR1 does. As there are multiple PKC isoforms in conjunctival goblet cells some of which are Ca 2+ -dependent and Ca 2+ -independent PKC isoforms, MaR2 and MaR1 may be activating different PKC isoforms to stimulate secretion [42]. Identification of a MaR2-specific receptor and a more detailed investigation of the components of the signaling pathways could clarify some of the differences between MaR2 and MaR1 and their use of signaling pathways in conjunctival goblet cells.
A common type of chronic inflammation on the ocular surface is ocular allergy, a disease initiated by an allergic stimulus. Inflammatory ocular diseases usually cause hypersecretion of mucins. One of the central stimulatory mediators causing hypersecretion in allergic diseases is histamine [7]. When rat conjunctival goblet cells are preincubated with MaR2 before stimulation with histamine, the increase in both [Ca 2+ ] i and secretion decrease. This suggests that MaR2 can block the inflammatory effect of histamine on goblet cells decreasing mucin secretion. Many other SPMs, including LXA 4 , RvD1, RvE1, and MaR1, similarly counter-regulate the effect of histamine in cultured rat conjunctival goblet cells [15,16,43,44]. Ours findings herein support a role of MaR2, in homeostasis by stimulating goblet cell secretion in health and decreasing overproduction in diseases such as ocular allergy.
Information about the function of MaR2 in disease in other organs is limited. MaR2 limits polymorphonuclear neutrophil (PMN) entry during inflammation and stimulates phagocytosis, similar to the actions of MaR1 [11,12]. Furthermore, the anti-inflammatory, pro-resolving, and anti-atherosclerotic effects of MaR2 might be beneficial in diseases such as myocardial infarction and acute and chronic heart failure [11,45]. MaR2 is likely to be active in many additional diseases and tissues.
We conclude that MaR2 and MaR1 stimulate conjunctival goblet cell function especially secretion, by activating different, but overlapping GPCR and signaling pathways, and furthermore counter-regulate histamine stimulated increase in [Ca 2+ ] i . Thus, MaR2 and MaR1 play a role in maintaining the ocular surface and tear film homeostasis in health and disease. As MaR2 and MaR1 each modulate conjunctival goblet cell function, they

Measurement of High Molecular Weight Glycoconjugate Secretion
Cultured rat conjunctival goblet cells were trypsinized and transferred to 24 well plates. The cells were serum starved in free RPMI 1640 media containing 0.5% bovine serum album (BSA) for 120 min. MaR2 (10 −10 -10 −8 M) was then added alone or the cells were incubated with an inhibitor for 30 min, and then stimulated with MaR2 (10 −10 -10 −8 M) or carbachol (10 −4 M) for 2 h. The amount of goblet cell high molecular weight glycoconjugate secretion was measured using the lectin UEA-1 in an enzyme linked lectin assay (ELLA). Glycoconjugate secretion is shown as -fold increase above basal (which was set to 1).

Statistical Analysis
Data are expressed as mean ± SEM. N indicates cells cultured from different animals. Data were analyzed by either Student's t-test or one-way ANOVA followed by Tukey test. p < 0.05 was considered significant. Statistical analyses were performed using Excel (version 16.16.27,Microsoft Corp) and GraphPad Prism (version 9.3.1).

Conclusions
As MaR2 and MaR1 each modulate conjunctival goblet cell function, they each have potential as novel, but differing, options for treatment of ocular surface inflammatory diseases including allergic conjunctivitis and dry eye disease.